This journal is c the Owner Societies 2011 Phys. Chem. Chem. Phys., 2011, 13, 6699–6709 6699 Cite this: Phys. Chem. Chem. Phys., 2011, 13, 6699–6709 Mechanisms and advancement of antifading agents for fluorescence microscopy and single-molecule spectroscopywz Thorben Cordes,* ab Andreas Maiser, c Christian Steinhauer, a Lothar Schermelleh* c and Philip Tinnefeld* ad Received 23rd September 2010, Accepted 14th January 2011 DOI: 10.1039/c0cp01919d Modern fluorescence microscopy applications go along with increasing demands for the employed fluorescent dyes. In this work, we compared antifading formulae utilizing a recently developed reducing and oxidizing system (ROXS) with commercial antifading agents. To systematically test fluorophore performance in fluorescence imaging of biological samples, we carried out photobleaching experiments using fixed cells labeled with various commonly used organic dyes, such as Alexa 488, Alexa 594, Alexa 647, Cy3B, ATTO 550, and ATTO 647N. Quantitative evaluation of (i) photostability, (ii) brightness, and (iii) storage stability of fluorophores in samples mounted in different antifades (AFs) reveal optimal combinations of dyes and AFs. Based on these results we provide guidance on which AF should preferably be used with a specific dye. Finally, we studied the antifading mechanisms of the commercial AFs using single-molecule spectroscopy and reveal that these empirically selected AFs exhibit similar properties to ROXS AFs. 1. Introduction Fluorescence light microscopy has become an indispensable tool in various scientific fields, ranging from biomedical research to material sciences. One of its key features is the possibility to specifically label and detect structural components of interest with spectrally distinct fluorophores, e.g., to analyze the spatial distribution of biomolecules within cells and tissues by immunohistochemistry. 1 More recently, the introduction of single-molecule approaches 2,3 and super-resolution imaging techniques, 4–7 have further extended the capabilities and range of applications. Modern fluorescence applications are strongly dependent on the performance and characteristics of the fluorescent probes used. Important properties of the labels are their brightness (given by the product of extinction coefficient at the excitation wavelength and the fluorescence quantum yield), photostability (i.e., resistance to irreversible, light-induced reactions), storage stability of stained samples, solubility in water (for biological applications), the capability to chemically link the label to the structure of interest and finally a minimized influence of the label onto the labeled structure itself. Organic fluorophores often represent the premier choice due to their brightness, chemical flexibility, small size and many new labeling strategies even for in vivo applications. 1,8 Many substances, especially reductants such as ascorbic acid (AA), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox, TX), 9,10 p-phenylenediamine (PPD, used in the commercial product Vectashield), 1,4-diazabicyclo[2.2.2]octane (DABCO, used in Ibidi-MM) 11 or n-propyl gallate and triplet-quenchers such as mercaptoethylamine 12 and cyclo- octatetraene, 12,13 have been known to improve the photo- stability of fluorophores for microscopy and single-molecule spectroscopy. 14,15 Antifading substances are typically dissolved in glycerol-buffer or aqueous solution, which preserve the sample morphology. It should be noted that other frequently used embedding media have polymerizing formulae (e.g., Prolong Gold, Moviol). While good antifading properties have been reported, 14,15 cells embedded in these hardening media show substantial flattening, which is often disadvantageous for applications where the preservation of the 3-dimensional (3D) morphology is a requirement. Herein, we compare a a Applied Physics – Biophysics & Center for NanoScience (CeNS), Ludwig Maximilian University of Munich, Amalienstr. 54, 80799 Munich, Germany. E-mail: [email protected], [email protected]; Fax: +49 531 391 5334 b Biological Physics Research Group, Department of Physics, University of Oxford, Clarendon Laboratory, Parks Road, Oxford OX1 3PU, United Kingdom c LMU Biocenter, Department of Biology, Ludwig Maximilian University of Munich, Grosshaderner Str. 2, 82152 Planegg-Martinsried, Germany. E-mail: [email protected]d NanoBioSciences, Institute of Physical and Theoretical Chemistry, TU Braunschweig, Hans-Sommer-Str. 10, 38106 Braunschweig, Germany w This article was submitted as part of a Themed Issue on single- molecule optical studies of soft and complex matter. Other papers on this topic can be found in issue 5 of vol. 13 (2011). This issue can be found from the PCCP homepage http://www.rsc.org/pccp. z Electronic supplementary information (ESI) available. See DOI: 10.1039/c0cp01919d PCCP Dynamic Article Links www.rsc.org/pccp PAPER Downloaded by Ludwig Maximilians Universitaet Muenchen on 25/04/2013 13:08:05. Published on 11 February 2011 on http://pubs.rsc.org | doi:10.1039/C0CP01919D View Article Online / Journal Homepage / Table of Contents for this issue
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This journal is c the Owner Societies 2011 Phys. Chem. Chem. Phys., 2011, 13, 6699–6709 6699
(DABCO, used in Ibidi-MM)11 or n-propyl gallate and
triplet-quenchers such as mercaptoethylamine12 and cyclo-
octatetraene,12,13 have been known to improve the photo-
stability of fluorophores for microscopy and single-molecule
spectroscopy.14,15 Antifading substances are typically dissolved
in glycerol-buffer or aqueous solution, which preserve the
sample morphology. It should be noted that other frequently
used embedding media have polymerizing formulae (e.g., Prolong
Gold, Moviol). While good antifading properties have been
reported,14,15 cells embedded in these hardening media show
substantial flattening, which is often disadvantageous for
applications where the preservation of the 3-dimensional
(3D) morphology is a requirement. Herein, we compare a
a Applied Physics – Biophysics & Center for NanoScience (CeNS),Ludwig Maximilian University of Munich, Amalienstr. 54,80799 Munich, Germany. E-mail: [email protected],[email protected]; Fax: +49 531 391 5334
b Biological Physics Research Group, Department of Physics,University of Oxford, Clarendon Laboratory, Parks Road,Oxford OX1 3PU, United Kingdom
cLMU Biocenter, Department of Biology, Ludwig MaximilianUniversity of Munich, Grosshaderner Str. 2,82152 Planegg-Martinsried, Germany.E-mail: [email protected]
dNanoBioSciences, Institute of Physical and Theoretical Chemistry,TU Braunschweig, Hans-Sommer-Str. 10, 38106 Braunschweig,Germany
w This article was submitted as part of a Themed Issue on single-molecule optical studies of soft and complex matter. Other papers onthis topic can be found in issue 5 of vol. 13 (2011). This issue can befound from the PCCP homepage http://www.rsc.org/pccp.z Electronic supplementary information (ESI) available. See DOI:10.1039/c0cp01919d
PCCP Dynamic Article Links
www.rsc.org/pccp PAPER
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View Article Online / Journal Homepage / Table of Contents for this issue
This journal is c the Owner Societies 2011 Phys. Chem. Chem. Phys., 2011, 13, 6699–6709 6703
reduced mobility lead to comparable conditions for ROXS
and commercial AFs as also in samples with enzymatic oxygen
removal the final oxygen concentration only reaches the lower
micromolar range.32
To compare the different AFs for immunofluorescence
we performed photobleaching experiments in a commercial
confocal microscope frequently used for biological studies
(UltraVIEW VoX, PerkinElmer, details see Materials and
Methods). Human HeLa cells grown on microscope coverslips
were immunolabeled with antibodies conjugated to different
fluorophores. Fluorophore-coupled antibodies against GFP
were used to label expressed GFP-tagged histone H2B-GFP
with an organic dye of choice (Cy3B, ATTO 550, ATTO
647N). Histone H2B-GFP is stably incorporated into nucleosomes
of chromatin and is evenly distributed within the nucleus.
Alternatively, histones were detected with primary mouse
monoclonal antibodies against Pan-histone and secondary
anti-mouse antibodies conjugated to the respective dyes
(Alexa 488, Alex 594, Alexa 647). Regions with cells were imaged
and subsequently bleached in iterative cycles; the average
fluorescence intensity of bleached regions was registered after
each cycle.
ATTO 647N as an example for carborhodamine dyes
Fig. 3 shows exemplary images of typical bleaching experi-
ments with the fluorophore ATTO 647N. This carborhodamine
type dye (structure see Scheme 1) is known for its outstanding
photophysical performance (emax = 150 000 l mol�1 cm�1,
ffl = 0.65 as stated on the ATTO-TEC homepage,
www.atto-tec.com) especially on the single-molecule level.33
Fluorescent nuclei of cells embedded in either PBS or
Vectashield show hardly any remaining fluorescence after
40 bleaching cycles reflecting ensemble bleaching of the fluoro-
phore (Fig. 3a/b). In contrast, when using ROXS (AA/MV) as
buffer medium only partial bleaching of ATTO 647N is
observed (Fig. 3c) indicating a stronger resistance against
photobleaching.
For quantitative evaluation the mean fluorescence intensity
over time was determined (details see Material and Methods).
On average for each buffer condition, 10 independent time
series (10–20 nuclei each) were recorded at least on two
different days (Fig. 4).
The reference bleaching behaviour of PBS is represented by
black squares in Fig. 4a; the intensity is reduced to E0.25
of its original value after E35 bleaching cycles (bc). The
experimental data is well reproduced by a bi-exponential decay
(solid lines in Fig. 4a). Results from repetitive experiments and
fitting of these data are summarized in Table S1; fit curves
from a representative experiment are shown in Fig. 4a together
with experimental data. According to these results, the
fluorophores bleach with two different bleaching com-
ponents and a mean bleaching lifetime tm = 23 � 2 bc with
tm = A1 � t1 + A2 � t2. A non-exponential behavior may be
attributed to differing microenvironments of the fluorophores
and has been reported before.15
VS shows a very similar relative bleaching lifetime of
1.3� 0.6 with respect to PBS but with a considerable amplitude
of a fast decay component and overall large experimental errors
between different measurements (Fig. 4a, circles; Table S1).y Asignificant increase of the photobleaching resistance of ATTO
647N is observed for Ibidi-MM, ROXS (AA/MV) and ROXS
(TX/TQ) with mean bleaching lifetimes over several experi-
ments of 2.3 � 0.2, 2.9 � 0.5 and 3.1 � 0.5 relative to PBS,
respectively (Table S1). In all cases the fraction of the fast
bleaching component is significantly reduced to E10%
(Fig. 4a). These data quantitatively evidence that both ROXS
buffers and Ibidi-MM are able to significantly enhance photo-
stability of ATTO 647N in fluorescence imaging.
In the next step we compare the mean brightness of
the fluorophores, i.e., of fresh samples (mounted shortly
before performing photobleaching experiments) and aged
samples (after 3 days storage at 4 1C) for the various media
Fig. 3 Confocal images of HeLa histone H2B-GFP expressing cells
labeled with ATTO 647N-coupled GFP antibodies. Samples were
embedded using different mounting media. Selected images of a time
series with (a) PBS (b) Vectashield and (c) ROXS (AA/MV) are shown
here exemplarily. The ATTO 647N dye was excited at 635 nm (close to
its absorption maximum at E644 nm). The panels show fluorescently
labelled nuclei before and after 40 iterative cycles of confocal imaging
and bleaching the entire field of view. Significantly less fluorescence
intensity is left in the PBS- and VS-embedded samples, whereas ROXS
effectively preserves fluorescence. Bar is 20 mm.
y Note that all relative bleaching constants in Table S1 and in the textare derived from several measurement days, while the data in theFig. 4a/5a/6a/S1a/S2/S3a) show representative mean bleaching curvesfrom a particular experimental day.
This journal is c the Owner Societies 2011 Phys. Chem. Chem. Phys., 2011, 13, 6699–6709 6705
in Fig. S1a and Table S1. We note a significant enhancement
of the brightness of ATTO 550 in Ibidi-MM compared to
other AFs. Upon storage in both ROXS media we observe a
similar drop in fluorescence as for Alexa 488 (Fig S1b).
Experiments with Alexa 594 also follow these described trends:
all AFs have a positive influence on the photobleaching
resistance of the dye with moderate increases by a factor of
1.5 � 0.1 for ROXS (AA/MV) with respect to PBS (Fig. S2,
Table S1). Both VS and ROXS (TX/TQ) show a signifi-
cant increase by factors of 2.5 and 2.9, respectively, while
Ibidi-MM performs outstandingly well for Alexa 594 with
only 15% fluorescence loss compared to PBS (Fig. S2a, note
that the data could not be fitted to a bi-exponential function).
Again a moderate increase of brightness is observed for
Ibidi-MM and for ROXS (AA/MV) shortly after mounting
while the fluorescence intensity seems to be better preserved by
VS and Ibidi-MM when storing the sample (Fig. S2b).
Cyanine dyes
In the next section we investigated the influence of AFs on the
photophysical properties of cyanine dyes. As a first example
we chose Cy3B, a fluorophore with an absorption- and emission-
maximum at 558 nm and 572 nm, respectively. This dye is
frequently used in single-molecule FRET studies.34,35
As already found for rhodamine dyes all AFs clearly
increase the resistance of the fluorophore Cy3B against photo-
bleaching (Fig. 6a).
The mean bleaching lifetime of PBS (Fig. 6a, squares) is
increased by factors between 1.6 and 2.1 for ROXS (AA/MV),
Ibidi-MM, VS and ROXS (TX/TQ), compare Fig. 6a and
Table S1.
For all AF media we observed a significant decrease in
brightness of the fluorophore—a result that is surprising
considering that ROXS (AA/MV) was shown to increase the
brightness of surface-immobilized or diffusing fluorophores in
Fig. 5 Photobleaching experiments of the dye Alexa 488. (a) Time-course of the normalized fluorescence intensity over E25 bleaching cycles for
different buffers: PBS, squares; VS, circles; Ibidi-MM, stars; ROXS (AA/MV), triangles; ROXS (TX/TQ), inverted triangles. Mean curves of at
least five measurements from one particular measurement day are shown. Standard deviations were typically below 0.02 and are thus omitted for
clarity. Bi-exponential fits are shown as solid lines in the color of the respective data set. Fit results are summarized in Table S1. (b) Relative
fluorescence intensity for the different AFs before photobleaching and for aged samples (day 0 = fresh sample; day 3 = sample aged for three
days). Error bars indicate the standard error of mean intensities.
Fig. 6 Photobleaching experiments of the dye Cy3B. (a) The graph shows the time-course of the normalized fluorescence intensity over
E35 bleaching cycles for different AFs: PBS, squares; VS, circles; Ibidi-MM, stars; ROXS (AA/MV), triangles; ROXS (TX/TQ), inverted
triangles. Mean curves of at least five measurements from one particular measurement day are shown. Standard deviations were typically below
0.02 and are thus omitted for clarity. Bi-exponential fits are shown as solid lines in the color of the respective data set. Fit results are summarized in
Table S1. (b) Relative fluorescence intensity for the different AFs before photobleaching and for different aging-stages (day 0 = fresh sample;
day 3 = sample aged for three days). Error bars indicate the standard error of mean intensities.
and 3-dimensional structured illuminationmicroscopy (3D-SIM)40
that are generally more demanding for the employed dyes. The
choice of the right antifade medium makes an important
contribution to the dye performances. This paper gives useful
guidelines for their application and provides a framework for
further optimization of antifading formulae of mounting
media on the basis of the ROXS principle.
Acknowledgements
The authors thank C. Eggeling for discussions and helpful
information. We thank U. Rothbauer (ChromoTek GmbH)
for providing fluorophore coupled GFP antibodies. We are
grateful to S. Holzl for technical assistance. We are indebted to
H. Leonhardt for generous support. T. Cordes is supported
by a Marie-Curie Intra-European Fellowship provided by
the European Commission under the Seventh Framework
Programme (grant PIEF-GA-2009-255075). The project was
supported by the DFG (Inst 86/1051-1 to P. Tinnefeld and
SFB TR5 to L. Schermelleh), the Nanosystems Initiative
Munich (NIM) and the BioImaging Network Munich.
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